Streptavidin coated biosensor tips

Binding affinities between Stx1a(1–265) or Stx1a(25–265) and Munc18a or Munc18a(S306E/S313E) were measured using bio-layer interferometry in an Octet QK system equipped with Streptavidin-coated biosensor tips (ForteBio, Menlo Park, CA). Analyses were performed in 20 mm HEPES, pH 7.5, 90 mm NaCl, 20 μm EGTA, 0.1% β-mercaptoethanol, 0.05 mg/ml BSA at 30 °C using 96-well microplates (Greiner) shaking with a speed of 1,000 rpm during all experimental steps. Streptavidin biosensor tips were loaded over 10–30 s or up to a binding height of ∼1 nm in Stx1a(1–265) or Stx1a(25–265) containing a C-terminal Avitag sequence. To monitor complex formation between Munc18a and the syntaxin-1A constructs, the syntaxin-1A-loaded tips were dipped into a Munc18a dilution series (0, 5, 10, 20, 40, 80, 160, and 320 nm for WT Munc18a and Munc18a(S306E/S313E)) and allowed to associate for 200–1,000 s. Dissociation (200–1,000 s) was performed in buffer (20 mm HEPES, pH 7.5, 90 mm NaCl, 20 μm EGTA, 0.1% β-mercaptoethanol, 0.05 mg/ml BSA).

Biolayer interferometry was performed by using an Octet QK instrument (ForteBio, Menlo Park, CA). Biotinylated Her2-ECD (4 μg/mL in PBS, pH 7.4) was loaded onto streptavidin-coated Biosensor tips (ForteBio) for 10 min. Subsequently, unspecific binding was blocked by incubating the tips in PBS/BSA, pH 7.4, for 10 min. After measuring the baseline in PBS, pH 7.4 or 6.0, for 10 min, association with Fcab protein at indicated concentrations in PBS, pH 7.4 or 6.0, was monitored, followed by measuring the dissociation, again in PBS at pH 7.4 or 6.0.
Alternatively, Protein A-coated Biosensor tips (ForteBio) were loaded with 200 nM H10-03-6 (in PBS), followed by blocking with PBS/BSA and baseline-measurement (PBS). Finally, association was measured by incubation with indicated concentrations of soluble Her2-ECD in PBS, followed by detection of dissociation in PBS only. For this assay, all incubations were done at pH 7.4.
Interaction between Fc mutants and the neonatal Fc receptor (FcRn) was analyzed by surface plasmon resonance (SPR) by using a BIAcore 3000 instrument (GE Healthcare) as described previously [9 (link)]. Briefly, FcRn, which had been expressed in High Five cells and coated onto a CM5 chip, was incubated with 10 μg/mL Fc mutants in PBS, pH 6.0. The pH-dependent dissociation of Fc protein from FcRn was monitored by incubating the chip in PBS, pH 6.0, followed by PBS, pH 7.4.

Binding measurements were carried out at room temperature using an Octet RED96 system and streptavidin-coated biosensor tips (Pall ForteBio). The biotinylated operator DNAs were diluted to 200 nM in phosphate-buffered saline (PBS) with 0.2% bovine serum albumin (BSA) and 0.05% Tween-20 (PBS-T), pH 8.0, to be used as the antigen. Antigen-bound streptavidin tips were washed in PBS-T, pH 8.0 and dipped in LacI solutions in microplate wells with concentrations ranging from 0 to 40 nM in the same buffer during an association period. Tips were then returned to the washing well during a dissociation period. Experiments were carried out in technical triplicate, duplicate, and singlicate for −IPTG, lacO1 (natural operator), −IPTG, synthetic operator, and +IPTG, lacO1 conditions, respectively, at 10 LacI concentrations. Raw data were fit to 1:1 binding curves in Octet Data Analysis HT software version 10.0 using curve-fitting kinetic analysis with global fitting.